A rat basophil leukemic cell line (RBL-2H3) exhibited phosphorylation of both the 200,000 and 20,000-dalton chains of myosin following antigenic stimulation. Cells were primed with antigen specific IgE and labeled with (32P) orthophosphate and (35S)-methionine and then stimulated with antigen. Cells were then disrupted and myosin was isolated by immunoprecipitation with anti- platelet myosin antibodies. Upon stimulation, the amount of (32P)phosphate incorporated into the myosin chains increased from 0.4 to 0.8 moles/mole of light chain of myosin and from 0.8 to 1.6 moles/mole of the heavy chain of myosin. Most of this additional phosphorylation was accounted for by the incorporation of the phosphates into sites or both chains that are known to be phosphorylated by protein kinase C, although at the onset of antigen-induced secretion a transient increase in phosphorylation was observed at a site targeted by calmodulin-dependent light chain myosin kinase. The phosphorylation of the protein kinase C sites was more sustained. The subsequent dephosphorylation of these sites (after 10 to 15 min) occurred as the secretory response subsided. Additional studies revealed that both unphosphorylated and monophosphorylated forms of the light chain of myosin were phosphorylated by protein kinase C. The unphosphorylated form was also phosphorylated by the myosin kinase and the levels of the monophosphorylated form were thereby maintained at constant levels. The net effect was the de novo generation of the diphosphorylated form of the